The translocation of protons in biomolecular systems is a phenomenon of fundamental importance to such biological processes as ATP synthesis, enzyme catalysis, the maintenance of pH gradients, proton pumping, and bioenergetics. From the computational point of view the modeling of proton translocation represents a particularly difficult challenge-most notably because of the many complex interactions involved, the fact that bonding topologies are continually evolving due to the Grotthuss proton shuttling between water molecules and also possibly by amino acids, the interplay of charge migration via proton shuttling and classical ion diffusion, and the overall structural complexity of the target biomolecular systems. In most instances, the primary question is the way in which proteins utilize the proton shuttling characteristics of hydrogen bonded water chains, as well as how specific molecular groups within the protein participate in the proton translocation process via electrostatic interactions and possibly even through direct participation in the proton shuttling mechanism itself. In this project the continued development and application of a unique and powerful multiscale computer simulation methodology is described for the study of proton transport in several key classes of proton translocating biomolecular systems, including pumps (cytochrome c oxidase) enzymes (carbonic anhydrase), channels (M2 proton channels of influenza A and B), and antiporters (ClC chloride/proton antiporters). The overall research plan is made possible by a novel reactive Molecular Dynamics simulation approach that allows for the study of explicit long range proton transport through water molecules and ionizable molecular groups in hydrogen bonded networks, as well as by new innovations in enhanced free energy sampling methods, coarse-graining, and kinetic network theory. A primary target in the research will be to reveal the underlying microscopic biomolecular interactions which influence proton translocation in the above mentioned systems, as well as the way in which structural and chemical modifications of the proteins can affect this important property. These studies will be carried out in collaboration with a number of leading experimentalists, while adding a new dimension to the field of biomolecular computer simulation as a whole. 1